The present invention relates to an agent for gene therapy of dilated cardiomyopathy, more particularly, a gene expression vector which is obtained by inserting a gene encoding a sarcoglycan into an adeno-associated virus vector.
Cardiomyopathy is one of the heart diseases which shows contraction dysfunction and electrophysiological dysfunction as symptoms, and includes a group of heart diseases which lead to a sever heart failure and a sudden death. Cardiomyopathy is classified into dilated cardiomyopathy and hypertrophied cardiomyopathy, and the study for revealing the causes of each cardiomyopathy has been made. In the case of dilated cardiomyopathy (DCM), in spite of progress in the therapy, the prognosis of the patients is still poor and cardiac transplantation is necessary in the deteriorated cases (V. V. Michels, et al., New Engl.J.Med. 326, 77 (1992); E. K. Kasper, et al., J.Am.Coll.Cardiol. 23, 586 (1994); M. Packer, et al., New Engl.J.Med. 334, 1349 (1996); M. Packer, et al. New Engl.J.Med. 335,1107 (1996); R. M. Graham, W. A. Owens, N.Engl.J.Med. 341, 1759 (1999)). Therefore, it is necessary to develop a novel method for therapy which can improve the patient""s mortality and morbidity. Animal model is useful for developing such a novel method for therapy.
Gene transfer will be promising for the therapy of some type of DCM which is caused by the gene deletion. It has been demonstrated that the deletion of xcex4-sarcoglycan (xcex4-SG) gene is the cause of DCM in hamsters (A. Sakamoto, et al., Proc.Natl.Sci.Acad.U.S.A. 94, 13873 (1997); V. Nigro, et al., Hum.Mol.Genet. 6, 601 (1997)). Also, it has been found that the breakpoint of xcex4-SG gene in TO-2 hamster which is a model animal of DCM is present in the first intron, and large region including its promoter and the first exon is deleted in TO-2 hamster (A. Sakamoto, et al., Proc.Natl.Sci.Acad.U.S.A. 94, 13873 (1997)). Furthermore, dystrophin-associated glycoprotein complex (DAGC) links intracellular contractile machinery with extracellular matrix (G. F. Cox, L. M. Kunkel, Curr.Opin.Cardiol. 12, 329 (1997); K. H. Holt, et al., Mol. Cell 1, 841 (1998); M. D. Henry, K. P. Campbell, Curr.Opin.Cell Biol. 11, 602 (1999)).
The xcex4-SG makes a complex with the other three SGs (xcex1-, xcex2-, xcex3-SG) and connects intracellular dystrophin with laminin-2 at the extracellular matrix via xcex1- and xcex2-dystroglycans (G. F. Cox, L. M. Kunkel, Curr.Opin.Cardiol. 12, 329 (1997); K. H. Holt, et al., Mol.Cell 1, 841 (1998); M. D. Henry, K. P. Campbell Curr.Opin. Cell Biol. 11, 602 (1999)). In the myocardium of TO-2 hamster, the expressions of all of xcex1-, xcex2-, xcex3- and xcex4-SGs are missing in contrast with the myocardium of BIO 14.6 hamster (a model animal of hypertrophied cardiomyopathy) (T. Kawada, et al., F.E.B.S.Lett. 45, 405 (1999); T. Kawada, et al., Biochem.Biophys.Res.Commun. 259, 408 (1999)).
As one of the means for the therapy of heart diseases caused by the gene deletion or the change of gene expression in diseased cardiac muscle tissues such as dilated cardiomyopathy, molecular biological methods such as a direct transfer of gene into somatic cells of cardiac muscle tissues have been proposed.
Various methods have been proposed for gene transfer into somatic cells. For example, there have been proposed a gene transfer by DNA injection, a gene transfer using liposome, a gene transfer using a retrovirus vector, an adenovirus vector or an adeno-associated virus vector and the like. Important factors for the success of gene therapy include a high transfer efficiency, a stable expression of the gene, a tissue specificity, and a high safety to host.
For example, it has been proposed that a retrovirus vector is used for gene therapy. However, the use of retrovirus vector has many disadvantages. For example, virus DNA is randomly inserted into the chromosome of the host, and mutations may occur by such an insertion. In addition, the LTR (long terminal repeat) structure present in both ends of the retrovirus genome has a promoter/enhancer activity, and this activity may cause an activation of a gene locus which is adjacent to the inserted virus DNA.
As to those other than retrovirus, for example, adeno-associated virus (AAV) is studied as another system for delivering gene information into cells. The construction of a recombinant vector which comprises AAV DNA and heterogeneous gene under the control of AAV transcription promoter is described in, for example, U.S. Pat. No. 4,797,368 of Carter et al.
However, even in the case of using AAV vector, the gene therapy does not always succeed by satisfying various factors such as a high infection efficiency, a stable expression of the gene, a high tissue specificity, and a high safety. Especially, it has not been reported that the heart function of the patients of dilated cardiomyopathy can be improved by administering a gene expression vector into heart in vivo.
Thus, an object of the present is to construct a gene expression vector which can be used for a gene therapy of the patients of dilated cardiomyopathy.
Another object of the present invention is to construct a gene expression vector which can stably express a gene of interest when it is directly injected into the heart of the patients of dilated cardiomyopathy.
Still another object of the present invention is to construct a gene expression vector which can improve the heart function of the patients of dilated cardiomyopathy when it is directly injected into the heart of said patients.
Still another object of the present invention is to provide a pharmaceutical composition for gene therapy which comprises the aforementioned gene expression vector.
The present inventor has diligently studied to solve the aforementioned problems, and employed 5 week-old male TO-2 hamsters as a model animal (T. Kawada, et al., F.E.B.S.Lett. 45, 405 (1999); T. Kawada, et al., Biochem.Biophys.Res.Commun. 259, 408 (1999)), and employed a recombinant adeno-associated virus (rAAV) as a gene transfer vector, and injected xcex4-SG gene into the heart of TO-2 hamster in vivo. As a result, it has been found that the heart function is improved in TO-2 hamster where xcex4-SG gene has been transferred, and the present invention has been completed.
Thus, according to the present invention, there is provided a gene expression vector which is obtained by inserting a gene encoding sarcoglycan into an adeno-associated virus (AAV) vector.
Preferably, sarcoglycan is xcex4-sarcoglycan.
Preferably, the gene encoding sarcoglycan is a hamster or human sarcoglycan gene.
The gene expression vector of the present invention is preferably used for the therapy of heart diseases caused by deletion of sarcoglycan gene, particularly preferably dilated cardiomyopathy.
According to another aspect of the present invention, there is provided a pharmaceutical composition for gene therapy which comprises the gene expression vector of the present invention.
The pharmaceutical composition for gene therapy of the present invention is preferably used for the therapy of heart diseases caused by deletion of sarcoglycan gene, particularly preferably dilated cardiomyopathy.